A genetic analysis of the Gibraltar Neanderthals Lukas Bokelmanna,1, Mateja Hajdinjaka, Stéphane Peyrégnea, Selina Braceb, Elena Essela, Cesare de Filippoa, Isabelle Glockea, Steffi Grotea, Fabrizio Mafessonia, Sarah Nagela, Janet Kelsoa, Kay Prüfera, Benjamin Vernota, Ian Barnesb, Svante Pääboa,1,2, Matthias Meyera,2, and Chris Stringerb,1,2 aDepartment of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany; and bCentre for Human Evolution Research, Department of Earth Sciences, The Natural History Museum, London SW7 5BD, United Kingdom Contributed by Svante Pääbo, June 14, 2019 (sent for review March 22, 2019; reviewed by Roberto Macchiarelli and Eva-Maria Geigl) The Forbes’ Quarry and Devil’s Tower partial crania from Gibraltar geographic range from western Europe to western Asia (for an are among the first Neanderthal remains ever found. Here, we overview of all specimens, see SI Appendix, Table S1). Thus, show that small amounts of ancient DNA are preserved in the there is currently no evidence for the existence of substantial petrous bones of the 2 individuals despite unfavorable climatic genetic substructure in the Neanderthal population after ∼90 ka conditions. However, the endogenous Neanderthal DNA is present ago (4), the time at which the “Altai-like” Neanderthals in the among an overwhelming excess of recent human DNA. Using im- Altai had presumably been replaced by more “Vindija 33.19- proved DNA library construction methods that enrich for DNA like” Neanderthals (17). fragments carrying deaminated cytosine residues, we were able The Neanderthal fossils of Gibraltar are among the most to sequence 70 and 0.4 megabase pairs (Mbp) nuclear DNA of the prominent finds in the history of paleoanthropology. In 1848, a ’ ’ Forbes Quarry and Devil s Tower specimens, respectively, as well partial cranium (“Gibraltar 1”)wasfoundatForbes’ Quarry. Un- ’ as large parts of the mitochondrial genome of the Forbes Quarry surprisingly, in the historical context of its discovery, it was not individual. We confirm that the Forbes’ Quarry individual was a ’ immediately recognized as belonging to a distinct type of hominin female and the Devil s Tower individual a male. We also show that (18) (Fig. 1). A few years later, the discovery of the Feldhofer the Forbes’ Quarry individual is genetically more similar to the Neanderthal-type specimen sparked interest in and investigations of ∼120,000-y-old Neanderthals from Scladina Cave in Belgium (Scladina the Forbes’ Quarry cranium, which is assumed to have belonged to I-4A) and Hohlenstein-Stadel Cave in Germany, as well as to a ∼60,000- to 70,000-y-old Neanderthal from Russia (Mezmaiskaya 1), a female Neanderthal (19, 20). A form of benign bone overgrowth than to a ∼49,000-y-old Neanderthal from El Sidrón (El Sidrón 1253) on the interior of the cranium (endocranial hyperostosis) (19) in northern Spain and other younger Neanderthals from Europe and suggests that the individual died at a relatively advanced age. In western Asia. This suggests that the Forbes’ Quarry fossil predates 1926, an excavation of a nearby Mousterian rock shelter yielded the the latter Neanderthals. The preservation of archaic human DNA in parts of a partial second Neanderthal cranium and an associated the warm coastal climate of Gibraltar, close to the shores of Africa, mandible (“Gibraltar 2”),whichbelongtoachild(18)withanes- raises hopes for the future recovery of archaic human DNA from timated age of 3 to 5 y at time of death (21, 22). While the Devil’s regions in which climatic conditions are less than optimal for DNA Tower child was excavated more systematically, the exact archae- preservation. ological context of the Forbes’ Quarry specimen was not recorded. In the absence of direct chronological data for both fossils, no Gibraltar Neanderthal | Forbes’ Quarry | paleogenetics | ancient DNA | library preparation Significance he retrieval of complete (1, 2) and partial (3, 4) nuclear ge- The remains of 2 Neanderthals were found in Gibraltar: the Tnome sequences from Neanderthals has begun to provide first at Forbes’ Quarry in 1848 and the second at Devil’s Tower insights into their population history. Apart from the ∼430- in 1926. Since their discovery, present-day human DNA con- thousand-year (ka)-old Sima de los Huesos individuals, from whom tamination has accumulated in the specimens. By developing a only very little DNA has been sequenced (5), the genetically most DNA library preparation method that reduces modern con- divergent Neanderthal lineage known to date is represented by the tamination before sequencing, we were able to isolate enough ∼130-ka-old Altai Neanderthal (Denisova 5) from Denisova Cave endogenous DNA from the specimens to determine their sex in the Altai Mountains in Russia, which has yielded a high-quality and to infer that the Forbes’ Quarry Neanderthal is more sim- genome sequence (1) and is one of the eastern-most Neanderthal ilar to 60,000- to 120,000-y-old Neanderthal specimens in specimens found. All other Neanderthal individuals from which Europe and western Asia than to younger Neanderthals. The substantial parts of the nuclear genome have been sequenced (1, 3, laboratory protocols presented here improve access to ancient 4, 6) are more closely related to a >44-ka-old individual from DNA from specimens that are highly contaminated with Vindija Cave, Croatia (Vindija 33.19), the other individual from present-day human DNA. whom a high-quality genome sequence has been published (2). Author contributions: I.B., S.Pä., M.M., and C.S. designed research; L.B., M.H., S.B., E.E., These include the Hohlenstein-Stadel specimen from Southern I.G., and S.N. performed research; L.B., M.H., S.Pe., C.d.F., S.G., F.M., J.K., K.P., B.V., and Germany (4, 7, 8), as well as Scladina I-4A from Belgium (4, 9), M.M. analyzed data; and L.B. and M.M. wrote the paper. both of which are similar in age or slightly younger than the Altai Reviewers: R.M., Universite de Poitiers; and E.-M.G., CNRS and University Paris Diderot. ∼ Neanderthal (1, 4, 8); a 60- to 70-ka-old specimen from The authors declare no conflict of interest. ∼ Mezmaiskaya Cave, Russia (Mezmaiskaya 1) (1, 10); and a 80-ka- Published under the PNAS license. old specimen (Chagyrskaya 8 from Chagyrskaya Cave, Russia) (6, 11), Data deposition: The data reported in this paper have been deposited in the European which was also recently sequenced to high coverage (6). The ge- Nucleotide Archive (accession no. PRJEB31410). nomes of 4 late Neanderthals, which were dated to between 47 and 1To whom correspondence may be addressed. Email: [email protected], 39 ka [Goyet Q56-1 (12) and Spy 94a (13), both from Belgium; Les [email protected], or [email protected]. Cottés Z4-1514 (14) from France; and Mezmaiskaya 2 from Russia 2S.Pä., M.M., and C.S. contributed equally to this work. (15)] (3) are even more similar to Vindija 33.19 than those of the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. aforementioned individuals (Hohlenstein-Stadel, Chagyrskaya 8, 1073/pnas.1903984116/-/DCSupplemental. Mezmaiskaya 1, Scladina I-4A) (1, 3, 4, 16), despite spanning a Published online July 15, 2019. 15610–15615 | PNAS | July 30, 2019 | vol. 116 | no. 31 www.pnas.org/cgi/doi/10.1073/pnas.1903984116 A B Gibraltar Denisova Chagyrskaya Scladina Spy Goyet Hohlenstein-Stadel Les Cottés Vindija Mezmaiskaya El Sidrón Gibraltar Fig. 1. (A) Geographic locations of Gibraltar and other sites that are discussed in this study. (B) The Forbes’ Quarry cranium (Top) and the cranium and mandible of an infant found at Devil’s Tower (Bottom) on Gibraltar. Reprinted with permission from ref. 18. GENETICS consensus has been reached on the age of the specimens, which ancient DNA base damage resulting from the deamination of have been suggested to date to anywhere between marine isotope cytosine (C). This chemical reaction produces uracils (U), which stage 3 and 5 (∼30 to ∼130 ka ago) (23). It has been proposed that accumulate predominantly at the end of ancient DNA molecules Neanderthals inhabited Gibraltar until as late as 24,000 uncali- and are read by DNA polymerases as thymines (T) (38). Less brated radiocarbon years ago (24, 25), based on dates of charcoal than 5% of the sequences that start or end at a position at which from layers containing Mousterian artifacts. However, the accuracy the reference genome carries a C showed a T, suggesting that the of these dates has been questioned (26–29). vast majority, if not all, of these sequences derive from con- The Iberian Peninsula, and Gibraltar in particular, are located temporary human contamination (Fig. 2). on one of the extremes of the Neanderthal distribution and are To test whether small amounts of authentic ancient DNA thought to have served as a refuge for Neanderthals during could be present in these sequences, we computed the frequency glaciations (25, 30). In addition, it has been suggested that Ne- of C-to-T substitutions at the 5′-end for sequences that show a 3′ anderthals may have persisted in Gibraltar thousands of years C-to-T substitution, and vice versa (conditional substitution fre- after they were replaced by modern humans in other parts of quencies) (39). We found that among such sequences, C-to-T sub- Eurasia (24, 25). Genetic analysis of the Forbes’ Quarry and stitution frequencies were higher than 40% (Fig. 2). We thus Devil’s Tower specimens would help to determine their re- concluded that a population of highly deaminated ancient DNA latedness to other Neanderthals in western and central Eurasia. molecules is present in both specimens, but that these molecules are However, the warm Mediterranean climate in Gibraltar is un- present in a large excess of less deaminated recent human DNA.